ABSTRACT
Objective To investigate the roles and mechanisms of siRNA targeted lipocalin 2 (Lcn 2) gene silencing on hypoxia-induced cell apoptosis in retinal ganglion cells (RGC-5).Methods RGC-5 cells were subjected to hypoxic condition for various time duration (0 h,4 h,8 h,12 h,24 h,48 h),and quantitative real-time PCR and Western blot were used to evaluate the expression of Lcn 2 in mRNA and protein levels.Cells were divided into 4 groups:control group,hypoxia group,siNC + hypoxia group,in which cells were transfected with negative control for siRNA,and then subjected to hypoxic condition for 24 h,and siLcn 2 + hypoxia group,in which cells were transfected with Lcn 2 siRNA,and then subjected to hypoxic condition for 24 h).Next,cell apoptotic rate and Caspase-3 activity were detected using ELISA.The fluorescence intensity of reactive oxygen species (ROS) was assayed using DCFH-DA kit,and mitochondrial membrane potential assay kit was used to evaluate the mitochondrial membrane potential.Finally,the expression levels of cleaved-Caspase-3 (c-Caspase-3),cleaved-PARP (c-PARP),Bax,Bcl-2 and cytochrome C (Cyto C) were detected using Western blot.Results In the hypoxia group,Lcn 2 mRNA level at 4 h,8 h,12 h,24 h and 48 h was higher than that at 0 h (all P ＜0.05).Meanwhile,Lcn 2 protein level at 12 h,24 h and 48 h was also higher than that at 0 h (all P ＜ 0.05).Cell apoptotic rate in the hypoxia group (138.33％ ± 13.76％) was significantly higher than that in the control group (99.66％ ± 2.86％) (P ＜ 0.05),and siLcn 2 + hypoxia group (105.02％ ± 8.60％) was lower than siNC + hypoxia group (142.33％ ± 6.54％) (P ＜ 0.05).In addition,compared with the control group,the relative activity of Caspase-3 and the relative expression of c-Caspase-3 and c-PARP in the hypoxia group were all upregulated (all P ＜ 0.05);whereas the relative activity of Caspase-3 and the relative expression of c-Caspase-3 and c-PARP in the siLcn2 + hypoxia group were downregulated compared with the siNC + hypoxia group (all P ＜ 0.05).Moreover,the fluorescence intensity of ROS in the hypoxia group (4.26 0.63) was more enhanced than that in the control group (1.03 ± 0.11) (P ＜ 0.05),and the siLcn2 + hypoxia group (3.10 ± 0.24) was lower than siNC + hypoxia group (4.73 ± 0.26) (P ＜ 0.05).Furthermore,mitochondrial membrane potential,Cyto C expression and the relative ratio of Bax to Bcl-2 in the siLcn2 + hypoxia group was lower than those in the siNC + hypoxia group.Conclusion Lcn 2 Lcn2 gene silencing can inhibit cell apoptosis and ROS production induced by hypoxia,which may be achieved by suppressing mitochondrial apoptosis signaling pathway.